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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr4:74119706+74119805 100bp TGGTTGGTTCTTTCATGTGC GGATGAGCTGAAACGTCATAAAG
Mut= 91 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AAATTCTTTCATGTATCTCAAACCTAAACACAGCTTTGGGCAGATTGGCTTTTACGTGAGAGTGGAAAGTGTCAGAGGAGAAAATCTTCCTTGATCTGTGATCTGGAGAGTTGGAAGGGAAGGTGAGTTGTGATGTAGAGTGATGAGCTTCTTCTTGCTTTGGGTTGTATTGTACATTTGCTCTGGATATCCTGTTAAACATGTTTTTTGTTTTTGTCACCATCTTGAGCATTCTATTGGCTTCTTTCCTACAACCTCCTTCCCTTCCCTTCTGGTTGGTTCTTTCATGTGCTGAGGTTCCTATGTGTGTTTGCCTCTGTGatcttttgccgcttatctcatgccttttctttatgacgtttcagctcatccaccatacaccatgtgctttagagtgaaattctacccacatgaacccttgaagattaaagaagagctcacaaggtatttttcctttttgcatatttactccaccgcttgcattcaggtagagcctttgctatcttataggctagacattcctgttatcgatttgagaacacctccagGAAGTAGGCTGTGGAGTCAGCATTGTGTTCCTCATATTCTTGTTGAATTTGGGATACTTTATAGAGAACCTTGTTGAGAAAGTATTATCTGTACAAGAAAGCAATGGTTATAAAAGGGGAGCTCTCTAAAACATTTGACAATGGAATGCTAACAAAAGCAGAAGATGACTGTCTTTTGGGTATGGGCAAATGCAGAGAGCAGTCCTAGTCTGTCCTTTTGGGTCTAAGACTTTCTGGAAGAACAAGATTACTGGACAGAGAGGACAATTAGCTATTTTGCTTTCCACTTGCTACATGACAACTACCAAGCAGATGAACTCTCCAACTTTCTTCTCTTTCTCTCTCTCTC
This mutation is a 227 bp deletion beginning at Chromosome 4 position 74,119,755 bp and ending after 74,119,981 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48267 | TGG TTG GTT CTT TCA TGT GC | Common | A | |||
| 48268 | GGA TGA GCT GAA ACG TCA TAA AG | Wild type Reverse | A | |||
| 48269 | CAA GAA TAT GAG GAA CAC AAT GCT G | Mutant Reverse | A | |||
| 48270 | Fluorophore-1 | ATC TTT TGC CGC TTA TCT CAT G | Quencher-1 | WT Probe | ||
| 48271 | Fluorophore-2 | TCT GTG GAA GTA GGC TGT GGA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48267 | 0.40 uM |
| 48268 | 0.40 uM |
| 48269 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.